A basic technology in the field of molecular biology is the conversion of poly(A)+RNA (mRNA) to double-stranded (ds) complementary DNA (cDNA), which then can be inserted into a cloning vector for generating a cDNA library or expressing in an appropriate host cell. Advances in cDNA library construction technology have made possible the discovery and production of a wide range of biologically important proteins.
Main procedures for generating cDNA libraries which have been used during the last 15 years are comprehensively reviewed for example in Wu, ed. Methods in Enzymology (1987), vol. 152. For the most part, cDNA library construction technologies use poly(A)+RNA as a starting material. The intact poly(A)+RNA is characterized by a polyadenylated "tail" at its 3' end and a characteristic "CAP structure" at the 5' end. A critical requirement for cDNA library construction is to completely copy poly(A)+RNA to full-length cDNA and retain the complete sequence information on the structure of the protein encoded by mRNA. The "full-length" cDNA is therefore defined as a cDNA containing an entire sequence starting from a CAP site to the poly(A) tail.
One generalized and commonly used method (Gubler et al., 1983) by which the poly(A)+RNA is copied into cDNA employs reverse transcriptase, which starts at the 3' end of the mRNA from an oligo d(T) primer and proceeds towards the 5' end to generate a cDNA:mRNA hybrid. The RNA strand is then removed from the hybrid by action of RNase H and a second DNA strand is then synthesized using DNA polymerase I. The resulting heterogeneous mixture of double stranded cDNA (ds cDNA) molecules can then be cloned into recombinant DNA vector molecules using a variety of techniques. Unfortunately, this method does not allow synthesis of "full-length" cDNA because, for the majority of mRNAs, reverse transcriptase can not efficiently copy them into full-length cDNAs. The problem of "full-length" cDNA synthesis is more acute for long mRNAs as efficiency of copying is inversely proportional to the length of mRNA. Also, the current technology can generate deletions at the 5' and 3' ends of cDNA.
In an alternative approach (Okayama et al., 1982; see also Pruitt, International Patent, Appl. No. 89110816.9 of 14.06.89), poly(A) tails of mRNA molecules are first annealed to oligo (dT) linking with linearized vector DNA (vector primer). Then, the first strand of cDNA synthesized by reverse transcriptase is tailed at the 3' end by oligo dt which facilitates subsequent cloning by circularization into vector primer. This method also generate high level of cDNA clones containing truncated cDNAs due to non-full-length cDNA synthesis.
As a result, in conventional cDNA libraries, the majority of the cDNA clones do not have sequences close to the 5' end of the mRNAs. This results in a loss of important information required to make functional proteins. Two selection procedures have been developed in efforts to enrich cDNA libraries for "full-length" cDNA clones. In CAP retention procedure (CAPture) (Edery et al., 1995; Sonenberg et al., U.S. Pat. No. 5,219,989) cap-binding protein (eukaryotic initiation factor 4E) in combination with RNase A was used to purify the full-length cDNA:mRNA hybrid. Shorter duplexes corresponding to non-full-length cDNA fragments are not selected, since the cap is removed from the RNA moiety by nuclease treatment. Although the CAPture method could potentially enrich cDNA libraries for clones containing the authentic 5' ends, the yield of enriched full-length cDNA is very low, especially for long cDNAs (1-5%). The low yield can be a significant disadvantage for this technology.
In the "oligo-capping" method (Maruyama et a., 1994; Fromomt-Racine et al., 1993; Kato et al., International Patent, Publ. No. 0 625 572 A1, Appl. No. 93921061.3 of 22.09.93) the cap structure of mRNA is selectively replaced with an oligoribonucleotide, thus generating a chimeric oligonucleotide--full-length mRNA intermediates which are subsequently used for synthesis and cloning, preferably full-length cDNAs. However, this method is complicated, involving treatment of mRNA with an alkaline phosphatase, decapping mRNA with tobacco acid pyrophosphatase, and ligation of the oligonucleotide to the 5' end of mRNA by T4 RNA ligase. These multiple enzymatic steps degrade mRNA, thus generating incomplete cDNA fragments for subsequent cloning procedures. Size distribution of cDNA inserts in cDNA libraries generated by the "oligo-capping" method is less than 3 kb, which is much less than full-length mRNA size distribution (Kato et al., 1994) and indicates the low efficiency of "full-length" cDNA cloning by "oligo-capping" technology.
In summary, conventional methods for constructing of cDNA libraries containing the preferred full-length cDNA clones are restricted by low efficiency and the use of multiple, time-consuming steps. Accordingly, a simple method that would generate high quality full-length cDNA library is highly desirable.